Project Summary An single mutation (617G>A;Arg206His) within the kinase domain of just one of four human bone morphogenetic protein (BMP) receptors (ACVR1, ALK2) has recently been linked to the most catastrophic disorder of secondary (heterotopic) bone formation known to mankind. As a result of this identical substitution with histidine, all children presenting with features of classic FOP (Fibrodysplasia Ossificans Progressiva) eventually become encased in, and their movement blocked by, a second heterotopic skeleton. Because the cytoplasmic domains of the superfamily (TGF-2, transforming growth factor-2) of receptors are highly homologous, previously determined crystal structures of the TGF-2 type I receptor (T2RI) kinase domain in complex with the inhibitory protein FKBP12 and an ATP-analog small molecule inhibitor have lead to reliable structure-based homology models for interpreting the structural basis of altered BMP receptor kinase function in FOP. In this context, Arg206 appears to form a highly conserved ion pair with Asp269, situated in the glycine-serine rich (GS) regulatory region adjacent to the inhibitory protein (FKBP12) binding interface. Because the sidechain imidazole of histidine protonates and deprotonates within the physiological pH range, His-Asp ion pairs play unique roles in health and disease, acting as pH-sensitive switches for binding and release of O2 and CO2 by hemoglobin, and in a pH-sensitive molecular defect in the p53 tumor suppressor linked to a rare genetic disorder afflicting children in Brazil. Thus substitution with histidine, and only histidine, is predicted to induce a pH-sensitive conformational change in the protein backbone that leads to ligand-independent activation of the receptor under the hypoxic conditions of inflammation, postulated as an underlying trigger of episodes of explosive bone growth or "flare-ups" resulting from injuries to soft tissues. We will: (1) test this hypothetical pH-dependent mechanism of loss of autoinhibition by in vitro interaction analyses with the BMP kinase and FKBP12 by Surface Plasmon Resonance (SPR, BIAcore) assays, and (2) determine the crystal structures of the wildtype and Arg206His BMP kinases at physiological and hypoxic pH to identify precisely how the mutation perturbs the three- dimensional structure of the enzyme. These resulting structures will allow for optimization by rational design of a recently identified inhibitor (Dorsomorphin;a pyrrazolopyrimidine ATP-competitor) as a therapeutic drug targeted at the mutant kinase to block accumulation of extraskeletal bone in FOP, as well as the skeletal metastases of prostate cancer, the most common malignancy in men often resulting from stimulation of BMP receptor kinases.